Several criteria for diagnosis of alcohol use disorder (AUD) involve drinking despite negative consequences, meaning the decision to drink despite the loss of a job, damage to relationships, or increased risk of harm to oneself. One region of the brain that controls the decision to drink under conditions of risk is the insular cortex. Changes in neuronal function in the insula caused by excessive drinking may drive the transition to this risky, or aversion-resistant alcohol drinking. Our preliminary data indicates that specialized extracellular matrix structures, known as perineuronal nets (PNNs), may be involved in aversion-resistant drinking. PNNs in cortical regions primarily surround fast-spiking GABAergic interneurons that express the calcium-binding protein parvalbumin (PV). These neurons are important for cognition and tightly control the firing of excitatory cortical projection neurons. The overarching goals of this proposal are 1) to examine the cellular and molecular changes in PNNs on PV neurons in the insula after extended binge-like alcohol consumption by mice, and 2) to determine the behavioral consequences of manipulating PV neurons and PNNs on PV neurons in the insula on aversion-resistant drinking. In Specific Aim 1, we will examine the structure of PNNs at different time points after binge drinking using immunocytochemical methods, measure changes in the expression of PNN genes and proteins, and measure the activity of the proteases (MMP and ADAMTS metalloproteinase family) that regulate the remodeling of PNNs. In Specific Aim 2, we will measure changes in synaptic density and volume on PV neurons with and without PNNs after binge drinking using immunocytochemical methods and super-resolution microscopy. In addition, we will measure excitability and synaptic changes on these neurons after binge drinking using electrophysiological methods. Together, these two Specific Aims will provide important knowledge regarding the changes that occur in PNNs and their associated synapses after extended periods of binge alcohol drinking. In the third Specific Aim of this proposal, we will disrupt PNNs on PV neurons by knocking down the expression of two specific PNN proteins encoded by the genes Acan and Bcan using viral-delivered short hairpin RNAs. Viruses will be injected directly into the insula of mice expressing Cre recombinase in PV neurons (PV-Cre) for localized cell-type specific gene knockdown. These mice will be tested for consumption of ethanol alone and an aversive solution of ethanol containing quinine to determine the effect of PNN disruption on aversion-resistant drinking. Finally, we will directly manipulate the activity of insular PV neurons by viral delivery of designer receptors exclusively activated by designer drugs (DREADDs) to determine the role of these neurons in aversion-resistant ethanol consumption. These studies will provide information on the cellular and molecular changes that occur on PV neurons in the insula that drive the transition from binge drinking to high risk drinking and may lead to new therapeutic strategies to treat aversion-resistant drinking.